DETERMINATION OF p-AMINOBENZOIC ACID, CONJUGATED p [PDF]

Conjugated p-aminobenzoic acid may be determined after the same hy- drolysis procedure as that used by Marshall for sulf

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DETERMINATION p-AMINOBENZOIC

OF p-AMINOBENZOIC ACID, CONJUGATED ACID, AND p-NITROBENZOIC ACID IN BLOOD BY H. WILLIAM

(From the Division of Laboratories

ECKERT

and Research, New York State Department of Health, Albany)

(Received for publication,

November

25, 1942)

HeN 0 p-Aminobenzoic

COOH acid

J&N

0

SOzNHz

Sulfanilamide

Of several methods in use for the determination of sulfanilamide and related compounds perhaps the best is that of Marshall (11-13). It consists in diazotizing a trichloroacetic acid blood filtrate for 2 or 3 minutes with 0.1 per cent sodium nitrite, destroying excess nitrous acid with 0.5 per cent ammonium sulfamate solution containing KHzPOl as a buffer, and finally developing a red color suitable for calorimetric determination by coupling the diazotized solution with dimethyl-a-naphthylamine.’ A modification of this method has been found very satisfactory for the determination of p-aminobenzoic acid. The changes are briefly: (1) the concentration of sodium nitrite is increased from 0.1 to 0.2 per cent; (2) the concentration of ammonium sulfamate is increased from 0.5 to 2.0 per cent; (3) no buffer is used; and (4) the diazotization period is lengthened. Conjugated p-aminobenzoic acid may be determined after the same hydrolysis procedure as that used by Marshall for sulfanilamide, since both compounds in the conjugated form are acetylated at the amino group (14). Studies in this laboratory have dealt with the pharmacologic properties of p-nitrobenzoic acid administered to animals (15) and with its effects when 1 The later reagent of Bratton and Marshall (13) possesses certain advantages over dimethyl-a-naphthylamine and can be used equally well in all determinations in which the dimethyl-cr-naphthylamine is used in this paper. At the time of this investigation all the experimental work was carried out with the dimethyl-cy-naphthylamine reagent. Several preparations of the new reagent obtained at this time proved to be of unsatisfactory quality and it was not until after this work was completed that a high quality commercial product appeared on the market. 197

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Numerous recent investigations (l-10) have stressed the biologic importance of p-aminobenzoic acid, and it is therefore desirable to have a chemical method for determining this substance in biologic fluids. The structural relationship between sulfanilamide and p-aminobenzoic acid is .evident from the formulae.

198

DETERMINATION

OF

P-AMINORENZOIC

ACID

Procedure Preparation of Blood Filtrates-Measure 2 ml. of oxalated or titrated blood into 50 ml. flasks, add 30 ml. of distilled water, and mix thoroughly.

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introduced into cultures of various microorganisms (5, 16). A method for the determination of the nitro compound in biologic fluids was therefore required. The method here described consists in reducing the p-nitrobeneoic acid to p-aminobenzoic acid which is then determined by the modified Marshall method. The reduction of the nitro group to the amino group takes place rather easily, but not all methods of reduction are satisfactory for this analysis. Some of the reagents used were zinc and hydrochloric acid, stannous chloride, tin and hydrochloric acid, iron and hydrochloric acid; none of these proved sufficiently reliable and some of them interfered with the final analysis. Titanous chloride proved to be an excellent reducing agent. ApparatusA Klett-Summerson photoelectric calorimeter with green filter No. 56 and matched test-tubes of 12.5 mm. diameter, 15 by 150 mm., lipped Pyrex test-tubes graduated at 10 ml. and 12 ml. ReagentsSodium nitrite, 0.2 per cent, freshly prepared. Hydrochloric acid, approximately 4 IV. Ammonium sulfamate (LaMotte), 2 per cent. This is a much stronger solution than is necessary, but assures complete destruction of excess nitrite during the analysis. Dimethyl-a-naphthylamine (Eastman),1 ml. in 250 ml. of 95 per cent alcohol. Titanous chloride (LaMotte), 20 per cent solution, standardized. The latter is Trichloroacetic acid (Merck), 15 and 2.7 per cent solutions. conveniently prepared by diluting 18 ml. of the 15 per cent solution to 100 ml. p-Aminobenzoic acid and p-nitrobenzoic acid (Eastman) solutions. Each contains 100 mg. of the solute in 100 ml. of 2.7 per cent trichloroacetic acid. From these stock solutions dilutions are made for standards or recovery experiments in blood. Eastman chemicals are used after being recrystallized from water. Sulfanilamide (Winthrop), 100 mg. of sulfanilamide in 100 ml. of the dilute trichloroacetic acid. Add 36 gm. of tartaric acid (EastTartaric-hydrochloric acid mixture. man) and 42 ml. of concentrated hydrochloric acid to 100 ml. of distilled This solution has a slight yellow color which does not interfere with water. the test.

H.

W.

ECKERT

199

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Allow laking to take place for 5 minutes. Add 8 ml. of 15 per cent trichloroacetic acid slowly with rotation. Shake the mixture vigorously and allow to stand for 15 minutes. Filter through filter paper. The filtrate is used for determination of p-aminobenzoic acid, p-acetylaminobenzoic acid, and p-nitrobenzoic acid. Determination of p-Aminobenxoic Acid in Blood Filtrates-Place 10 ml. of the blood filtrate in a 50 ml. flask and add 2 ml. of water. Add 1 ml. of 0.1 per cent sodium nitrite solution and allow the mixture to stand for from 15 to 20 minutes. Then add 1 ml. of 2 per cent ammonium sulfamate, mix well, and let stand for from 2 to 3 minutes. Finally, add 5 ml. of the alcoholic dimethyl-a-naphthylamine and allow to stand for from 30 to 60 minutes in order to develop the maximum color. Read in the calorimeter with the blank set at zero. The blank for this determination, and also that for the conjugated (acetylated) p-aminobenzoic acid, consists of 10 ml. of 2.7 per cent trichloroacetic acid solution treated exactly as above. Determination of Conjugated (Acetylated) p-Aminobenzoic AcidPlace 10 ml. of the blood filtrate in a graduated Pyrex test-tube and add 0.5 ml. of acid. Place the tube in a boiling water bath for 1 hour, 4 N hydrochloric cool to room temperature, and dilute the mixture with water to the 10 ml. mark. Transfer to a 50 ml. flask and rinse the tube with 2 ml. of water, adding this rinsing to the first solution. The solution is now treated exactly as in the case of the free p-aminobenzoic acid. Determination of p-Nitrobenxoic Acid in Blood IMtrates-Place 10 ml. of the blood filtrate in a graduated Pyrex test-tube and add 1 ml. of tartarichydrochloric acid mixture and 2 drops of 20 per cent titanous chloride. The function of the tartaric-hydrochloric acid mixture is to prevent the formation of insoluble titanic acid on heating. Mix the contents of the tube well by rotating and then place in a boiling water bath for from 10 to 15 minutes. Cool the tube rapidly to room temperature and dilute the mixture with water to the 12 ml. mark. Transfer to a 50 ml. flask. Add 1 ml. of 0.2 per cent sodium nitrite solution, mix, and let stand for 20 minutes wit,h occasional swirling. This last step removes the remaining traces of titanous ion and also diazotizes the amine. Add 1 ml. of 2 per cent ammonium sulfamate, mix, and allow to stand for from 2 to 3 minutes. Finally, add 5 ml, of alcoholic dimethyl-a-naphthylamine and allow the color to develop from 4 to 5 hours before reading. This longer time is necessary for maximum color development, since the rate of coupling is greatly reduced in the presence of the additional reagents. The mixture may stand overnight at this point, without danger of fading. If the colored solution is not clear, centrifuge. Read in the calorimeter against a blank set at zero. The blank is 10 ml. of 2.7 per cent trichloroacetic acid solution treated exactly as is the sample.

200

DETERMINATION

OF

P-AMINOBENZOIC

rZCID

Standards-A standard containing 0.025 mg. of p-aminobenxoic acid or an equivalent amount of sulfanilamide may be used. It is treated exactly as described under the determination of p-aminobenzoic acid, Sulfanilamide is a convenient standard, since in solution it keeps indefinitely in a cool dark place. Calculations

EXPERIMENTAL

Conformity of Color Formation to Beer’s Law-Color production by paminobenzoic acid was found to follow Beer’s law. Similar experiments were performed with sulfanilamide and sodium p-aminobenzoate (Fig. 1). The factors are directly proportional to the molecular weights of the compounds over the concentration range 0.0025 to 0.05 mg. per 100 ml. Recovery of p-Aminobenzoic Acid from Blood-Recoveries of p-aminobenzoic acid added to horse blood in concentrations of 20, 10, 5, 2.5, and 1.25 mg. per cent were 96, 99, 96, 96, and 103 per cent respectively, average 98 per cent. Thus, the recovery of p-aminobenzoic acid from blood is of the sameorder as that of sulfanilamide which, at this dilution, is around 95 per cent. No attempts were made at this time to determine the percentage recovery of p-acetylaminobenzoic acid, for there is little reason to assume any difference in the ease of hydrolysis of this compound and acetylated sulfanilamide. Experiments showed that conjugated p-aminobenzoic acid in blood filtrates is completely hydrolyzed in a boiling water bath in 1 hour or less. Blood filtrates diluted 1:20 and 1:40 were analyzed for conjugated p-aminobenzoic acid and no significant difference was found in the results at the two dilutions. This indicates that coprecipitation with proteins is probably not an important source of error.

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With the Klett-Summerson photoelectric calorimeter, readings are directly proportional to the concentration of the substance being determined: the factor equals the amount of standard divided by the scale reading. To calculate free p-aminobenzoic acid, the scale reading is multiplied by the factor, with suitable corrections for the relative molecular Conjugated p-aminoweights if sulfanilamide was used as the standard. benzoic acid is determined by comparing the amounts found before and after hydrolysis. In determination of p-nitrobenzoic acid, the amounts of free and conjugated p-aminobenzoic acid are subtracted from the total amount found after reduction with titanous chloride. The difference, representing paminobenzoic acid derived from p-nitrobenzoic acid, is converted to the equivalent weight of p-nitrobenzoic acid, and the value so found is divided by 0.788 to correct for the fact that recovery was only 78.8 per cent of the theory in horse blood filtrates, as is described below.

H.

W.

201

ECKERT

Reduction of p-Nitrobenxoic Acid to p-Aminobenxoic Acid-Reduction of p-nitrobenzoic acid is quantitative (99 per cent) in dilute trichloroacetic acid corresponding to a 1: 20 blood filtrate (Table I). 2 drops of 20 per cent titanous chloride were found ample for the reduction. Table I shows that at the end of 15 minutes heating in a boiling water bath the reduction is quantitative, but after longer periods there is a considerable loss. For ex-

of color

TABLE I Acid in Dilute Trichloroacetic Acid Solutions Reduction of p-Nitrobenzoic Corresponding to Concentration of I:90 Blood Filtrate Time of heating for reduction p-Nitrobenzoic

acid 15 min.

mg. per cent

per cent

12.5 6.25 3.13 1.56 0.78

98 99

100 102

I

60 min.

percent 87 89 88 92 95

ample, after the reduction mixture is heated for 1 hour, the recovery of paminobenzoic acid is only 90 per cent of the theoretical. Heating at a lower temperature during the reduction also gave low results, unless the heating was continued for a much longer time. Samples reduced in a boiling water bath and then left at room temperature for several hours before further treatment sometimes gave very low values. Therefore, it is important to

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SCALE READINGS (Klett-~ummerson Photoelectric Calorimeter ) 1. Conformity of color formation to Beer’s law and relationship intensities. FIG.

202

DETERMINATION

OF

P-AMINOBENZOIC

ACID

II

TABLE

Recoveries _~~

Sample

heated Theory mg. *e* cent

20.0 10.0 5.0 2.5 1.25

of p-Nitrobenxoic

10 to 15 minutes

Acid

at 90-100”. FOUlld mg. $Jer

15.4 7.87 3.94 1.97 1.01

from

Horse

Blood

-Recovery

cent

per cent 77.0 78.7 78.8 78.8 SO.8 78.8

recoveries were obtained when widely varying amounts of p-nitrobenzoic acid were added to a given amount of blood (Table II). It does not seem probable, therefore, that the low results are due to coprecipitation. The results shown in Table II are typical and indicat>e that a correction factor of 0.788 must be used in calculating the amount of p-nitrobenzoic acid in blood. Acid-Solutions containing Efect of Titanous Chloride on p-Aminobenzoic 10 mg. per cent of p-aminobenzoic acid mere treated exactly as in the procedure for determining p-nitrobenzoic acid. At the end of 15 minutes heating, the loss was less than 3 per cent; at the end of 1 hour, it was nearly 15 per cent,. This accounts for some of the losses on continued heating, but it does not explain the great loss in recoveries of p-nitrobenzoic acid from blood filtrates.

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proceed with the diazotization immediately after rapid cooling of the reduction mixture. Recoveries of p-Nitrobenxoic Acid Added to Horse Blood--Recoveries of p-nitrobenzoic acid from horse blood were only about 78.8 per cent. The reason for this low recovery could not be determined. If heating is continued for 1 hour, the recovery drops to around 72.5 per cent; after 10 minutes at 65-75”, the recovery is only about 16 per cent of the amount theoretically present. However, the results seem t,o be very consistent, since practically the same percentage recoveries were obtained in blood samples from four different horses, including some 60 specimens covering the range 1.25 to 20.0 mg. per cent. The analysis was carried out at different protein concentrations by varying the dilution of the blood sample from 1: 20 to 1: 100, but the percentage recovery was the same in the presence of the different amounts of blood protein. Moreover, the same percentage

H.

W.

203

ECKERT

SUMXARY

A method is present,ed for the determination of p-aminobenzoic acid, p-acetylaminobenzoic acid, and p-nitrobenzoic acid in blood or other biologic fluids. The author wishes to express his appreciation gestions of Dr. Mary C. Pangborn. 2 Celitc

analytical

filter

aid

(Johns-Manville).

of the criticisms

and sug-

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Determination of p-Aminobenzoic Acid in Media-For the determination of p-aminobenzoic acid in broth cultures, the cultures are passed through a porcelain filter and 2 ml. samples of the filtrate mixed with 8 ml. of distilled water and 2 ml. of 15 per cent trichloroacetic acid. The determination is carried out as described under blood filtrates. The sample is centrifuged until clear and is then ready to be read in the calorimeter with a suitable blank set at zero. The determination of p-nitrobenzoic acid in broths or other media presents special difficulties. If the material is cloudy and cannot be easily cleared by centrifuging, as in the case of pneumococcus cultures, it is treated as previously described and the color allowed to reach a maximum. Some Celite* or similar substance is then added to clarify the suspension and the colored solution is centrifuged until clear; the centrifuge tubes are capped to prevent evaporation. The colored solutions should not be filtered, since as much as 20 to 25 per cent of the color may remain on the paper. In the presence of the concentrations of phosphate ordinarily in culture media (0.1 to 0.2 per cent), the method for the determination of p-nitrobenzoic acid is not applicable; but the limiting concentration of phosphate was not determined. When colored broths are analyzed, a blank on the untreated broth must be determined. Xpecijicity and Xensitivity-Certain chemicals, such as phenol, cresol, m- and o-aminobenzoic acids, aniline, sulfanilamide compounds, and many other related substances give color reactions by this method but are generally not found in biologic fluids. It might be mentioned here that peptone broths sometimes give a slight color under the conditions of the method and this interference has been traced to the presence of tryptophane (17). Mood filtrates from normal animals give no reaction. The reaction described is extremely sensitive and 1 y of p-aminobenzoic acid can easily be determined in 10 ml. of filtrate. By suitable reduction of volumes a still greater sensitivity may be attained and it is possible to determine 0.1 y in 1 ml. of filtrate if special small calorimeter tubes are used.

204

DETERMINATION

OF

p-AMINOBENZOIC

ACID

BIBLIOGRAPHY

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1. Woods, D. D., Brit. J. I&q. Path., 21, 74 (1940). 2. Rubbo, S. D., and Gillespie, J. M., Nature, 146, 838 (1940). 3. Ansbacher, S., Proc. Sot. Exp. Biol. and Med., 44, 248 (1940); Science, 93, 164 (1941). 4. Selbie, F. R., Brit. J. Exp. Path., 21, 90 (1940). 5. Miller, J. K., J. Pharmacol. and Exp. Therup., 71, 14 (1941). 6. Strauss, E., Lowell, F. C., and Finland, M., J. C&z. Inv., 20, 189 (1941). 7. Martin, G. J., and Ansbacher, S., J. Biol. Chem., 136, 441 (1941). 8. Janeway, C. A., J. Am. Med. As-n., 116, 941 (1941). 9. Landy, M., and Wyeno, J., Proc. Xoc. Exp. Biol. and Med., 46,59 (1941). 10. Fildes, P., Lancet, 1, 955 (1940). 11. Marshall, E. K., Jr., J. Biol. Chem., 122, 263 (1937-38). 12. Marshall, E. K., Jr., and Litchfield, J. T., Jr., science, 88,85 (1938). 13. Bratton, A. C., and Marshall, E. K., Jr., J. Biol. Chem., 128, 537 (1939). 14. Harrow, B., Power, F. W., and Sherwin, C. P., Proc. Sot. Exp. Biol. and Med., 24, 422 (1926-27). 15. Miller, J. Ii., in Annual report of the Division of Laboratories and Research, New York State Department of Health, Albany, 9 (1940). 16. Miller, J. K., J. Pharmacol. and Ezp. Therap., 72, 354 (1941); J. Bact., 42, 133 (1941). 17. Eckert, H. W., J. Biol. Chem., 148, 205 (1943).

DETERMINATION OF p -AMINOBENZOIC ACID, CONJUGATED p-AMINOBENZOIC ACID, AND p -NITROBENZOIC ACID IN BLOOD H. William Eckert J. Biol. Chem. 1943, 148:197-204.

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